Selective autophagy of RIPosomes maintains innate immune homeostasis during bacterial infection.

The NOD1/2-RIPK2 is a key cytosolic signaling complex that activates NF-κB pro-inflammatory response against invading pathogens. However, uncontrolled NF-κB signaling can cause tissue damage leading to chronic diseases. The mechanisms by which the NODs-RIPK2-NF-κB innate immune axis is activated and resolved remain poorly understood. Here, we demonstrate that bacterial infection induces the formation of endogenous RIPK2 oligomers (RIPosomes) that are self-assembling entities that coat the bacteria to induce NF-κB response. Next, we show that autophagy proteins IRGM and p62/SQSTM1 physically interact with NOD1/2, RIPK2 and RIPosomes to promote their selective autophagy and limit NF-κB activation. IRGM suppresses RIPK2-dependent pro-inflammatory programs induced by Shigella and Salmonella. Consistently, the therapeutic inhibition of RIPK2 ameliorates Shigella infection- and DSS-induced gut inflammation in Irgm1 KO mice. This study identifies a unique mechanism where the innate immune proteins and autophagy machinery are recruited together to the bacteria for defense as well as for maintaining immune homeostasis.

YOD1 sustains NOD2-mediated protective signaling in colitis by stabilizing RIPK2.

Inflammatory bowel disease (IBD) is a disorder causing chronic inflammation in the gastrointestinal tract, and its pathophysiological mechanisms are still under investigation. Here, we find that mice deficient of YOD1, a deubiquitinating enzyme, are highly susceptible to dextran sulfate sodium (DSS)-induced colitis. The bone marrow transplantation experiment reveals that YOD1 derived from hematopoietic cells inhibits DSS colitis. Moreover, YOD1 exerts its protective role by promoting nucleotide-binding oligomerization domain 2 (NOD2)-mediated physiological inflammation in macrophages. Mechanistically, YOD1 inhibits the proteasomal degradation of receptor-interacting serine/threonine kinase 2 (RIPK2) by reducing its K48 polyubiquitination, thereby increasing RIPK2 abundance to enhance NOD2 signaling. Consistently, the protective function of muramyldipeptide, a NOD2 ligand, in experimental colitis is abolished in mice deficient of YOD1. Importantly, YOD1 is upregulated in colon-infiltrating macrophages in patients with colitis. Collectively, this study identifies YOD1 as a novel regulator of colitis.

Disruption of XIAP-RIP2 Association Blocks NOD2-Mediated Inflammatory Signaling.

Inflammatory responses mediated by NOD2 rely on RIP2 kinase and ubiquitin ligase XIAP for the activation of nuclear factor κB (NF-κB), mitogen-activated protein kinases (MAPKs), and cytokine production. Herein, we demonstrate that selective XIAP antagonism blocks NOD2-mediated inflammatory signaling and cytokine production by interfering with XIAP-RIP2 binding, which removes XIAP from its ubiquitination substrate RIP2. We also establish that the kinase activity of RIP2 is dispensable for NOD2 signaling. Rather, the conformation of the RIP2 kinase domain functions to regulate binding to the XIAP-BIR2 domain. Effective RIP2 kinase inhibitors block NOD2 signaling by disrupting RIP2-XIAP interaction. Finally, we identify NOD2 signaling and XIAP-dependent ubiquitination sites on RIP2 and show that mutating these lysine residues adversely affects NOD2 pathway signaling. Overall, these results reveal a critical role for the XIAP-RIP2 interaction in NOD2 inflammatory signaling and provide a molecular basis for the design of innovative therapeutic strategies based on XIAP antagonists and RIP2 kinase inhibitors.

XIAP promotes melanoma growth by inducing tumour neutrophil infiltration.

Elevated expression of the X-linked inhibitor of apoptosis protein (XIAP) has been frequently reported in malignant melanoma suggesting that XIAP renders apoptosis resistance and thereby supports melanoma progression. Independent of its anti-apoptotic function, XIAP mediates cellular inflammatory signalling and promotes immunity against bacterial infection. The pro-inflammatory function of XIAP has not yet been considered in cancer. By providing detailed in vitro analyses, utilising two independent mouse melanoma models and including human melanoma samples, we show here that XIAP is an important mediator of melanoma neutrophil infiltration. Neutrophils represent a major driver of melanoma progression and are increasingly considered as a valuable therapeutic target in solid cancer. Our data reveal that XIAP ubiquitylates RIPK2, involve TAB1/RIPK2 complex and induce the transcriptional up-regulation and secretion of chemokines such as IL8, that are responsible for intra-tumour neutrophil accumulation. Alteration of the XIAP-RIPK2-TAB1 inflammatory axis or the depletion of neutrophils in mice reduced melanoma growth. Our data shed new light on how XIAP contributes to tumour growth and provides important insights for novel XIAP targeting strategies in cancer.

Design, synthesis and evaluation of novel thieno[2,3d]pyrimidine derivatives as potent and specific RIPK2 inhibitors.

In human cells, receptor-interacting protein kinase 2 (RIPK2) is mainly known to mediate downstream enzymatic cascades from the nucleotide-binding oligomerization domain-containing receptors 1 and 2 (NOD1/2), which are regulators of pro-inflammatory signaling. Thus, the targeted inhibition of RIPK2 has been proposed as a pharmacological strategy for the treatment of a variety of pathologies, in particular inflammatory and autoimmune diseases. In this work, we designed and developed novel thieno[2,3d]pyrimidine derivatives, in order to explore their activity and selectivity as RIPK2 inhibitors. Primary in vitro evaluations of the new molecules against purified RIPKs (RIPK1-4) demonstrated outstanding inhibitory potency and selectivity for the enzyme RIPK2. Moreover, investigations for efficacy against the RIPK2-NOD1/2 signaling pathways, conducted in living cells, showed their potency could be tuned towards a low nanomolar range. This could be achieved by solely varying the substitutions at position 6 of the thieno[2,3d]pyrimidine scaffold. A subset of lead inhibitors were ultimately evaluated for selectivity against 58 human kinases other than RIPKs, displaying great specificities. We therefore obtained new inhibitors that might serve as starting point for the preparation of targeted tools, which could be useful to gain a better understanding of biological roles and clinical potential of RIPK2.

Insights into Molecular characterization of NOD1-RIPK interaction and Transcriptional Modulation in Response to LPS in Spotted Snakehead, Channa punctata (Bloch, 1793).

NOD1, plays a pivotal role in immune responses against bacterial as well as viral invasions. While the downstream signaling pathway of NOD1 is well understood in mammals, its characterization in lower vertebrates remains elusive. In present study, an effort was made to identify and characterize downstream signaling cascade of NOD1 in response to LPS, a potential ligand of NOD1 in teleosts, in spotted snakehead. In addition, the temporal effect of LPS on transcriptional modulation of NOD1 and its downstream signaling molecule RIPK2 was investigated. Docking studies revealed well conserved leucine rich domains of NOD1 that could bind with LPS. Further, NACHT-ATP interactions revealed differences in ATP binding motifs within the NACHT domain in spotted snakehead compared to those reported in other fish species and mammals pointing towards species-specific nature of ATP interactions within the NACHT domain. Further, it was revealed that the ssNOD1-CARD domain interacts with the CARD domain of downstream signaling molecule ssRIPK. Interestingly, LPS treatment modulated the expression of both, ssNOD1 and ssRIPK2 in a time-dependent manner.

RIPK2 promotes the progression of colon cancer by regulating BIRC3-mediated ubiquitination of IKBKG.

Colon cancer is a cancer with high morbidity and mortality worldwide. Receptor interacting serine/threonine kinase 2 (RIPK2) has been identified as a proto-oncogene, but its role in colon cancer is largely unknown. Herein, we found that RIPK2 interference could inhibit the proliferation and invasion of colon cancer cells, and promote apoptosis. Baculoviral IAP repeat containing 3 (BIRC3) is an E3 ubiquitin ligase, which was found highly expressed in colon cancer cells. Co-immunoprecipitation (Co-IP) experiments showed that RIPK2 could directly bind with BIRC3. Then, we demonstrated that RIPK2 overexpression promoted the expression of BIRC3, BIRC3 interference could eliminate RIPK2-dependent cell proliferation and invasion, and BIRC3 overexpression rescued the suppressive effect of RIPK2 interference on cell proliferation and invasion. We further identified IKBKG, an inhibitor of nuclear factor kappa B, as a ubiquitination substrate targeted by BIRC3. IKBKG interference could eliminate the inhibitory effect of BIRC3 interference on cell invasion. RIPK2 could promote BIRC3-mediated ubiquitination of IKBKG, inhibit the expression of IKBKG protein, and promote the expression of NF-κB subunits p50 and p65 proteins. In addition, DLD-1 cells transfected with sh-RIPK2 or/and sh-BIRC3 were injected into mice to establish a tumor xenograft model, and we found that administration of sh-RIPK2 or sh-BIRC3 impeded the growth of xenograft tumors in vivo, and co-administration displayed a better inhibitory effect. In general, RIPK2 promotes the progression of colon cancer by promoting BIRC3-mediated ubiquitination of IKBKG and activating the NF-κB signaling pathway.

RIPK protein kinase family: Atypical lives of typical kinases.

Receptor Interacting Protein Kinases (RIPKs) are a family of Ser/Thr/Tyr kinases whose functions, regulation and pathophysiologic roles have remained an enigma for a long time. In recent years, these proteins garnered significant interest due to their roles in regulating a variety of host defense functions including control of inflammatory gene expression, different forms of cell death, and cutaneous and intestinal barrier functions. In addition, there is accumulating evidence that while these kinases seemingly follow typical kinase blueprints, their functioning in cells can take forms that are atypical for protein kinases. Lastly, while these kinases generally belong to distinct areas of innate immune regulation, there are emerging overarching themes that may unify the functions of this kinase family. Our review seeks to discuss the biology of RIPKs, and how typical and atypical features of this family informs the activity of a rapidly growing repertoire of RIPK inhibitors.

RIPK2 NODs to XIAP and IBD.

The receptor-interacting protein kinases (RIPKs) are key regulators of inflammatory signalling and cell death pathways triggered by innate immune receptors, and RIPKs have emerged as promising therapeutic targets for treatment of immune-related disorders. RIPK2 mediates signalling responses initiated by the bacterial-sensing pattern recognition receptors nucleotide-binding oligomerization domain-containing proteins 1 and 2 (NOD1/2), which play a key role in regulation of intestinal immunity and inflammation. Modification of RIPK2 by non-degradative ubiquitin chains generated by the E3 ubiquitin ligase XIAP and other ligases govern NOD1/2 signalling. Recent advances suggest that the interaction between RIPK2 and XIAP is a druggable protein-protein interaction to modulate NOD1/2-dependent immune responses. Here, we discuss the mechanistic function of RIPK2 in immune signalling, its clinical relevance, and the on-going efforts to target RIPK2 in inflammatory bowel disease and beyond.

Receptor-interacting protein kinase 2 (RIPK2) profoundly contributes to post-stroke neuroinflammation and behavioral deficits with microglia as unique perpetrators.

BACKGROUND: Receptor-interacting protein kinase 2 (RIPK2) is a serine/threonine kinase whose activity propagates inflammatory signaling through its association with pattern recognition receptors (PRRs) and subsequent TAK1, NF-κB, and MAPK pathway activation. After stroke, dead and dying cells release a host of damage-associated molecular patterns (DAMPs) that activate PRRs and initiate a robust inflammatory response. We hypothesize that RIPK2 plays a damaging role in the progression of stroke injury by enhancing the neuroinflammatory response to stroke and that global genetic deletion or microglia-specific conditional deletion of Ripk2 will be protective following ischemic stroke. METHODS: Adult (3-6 months) male mice were subjected to 45 min of transient middle cerebral artery occlusion (tMCAO) followed by 24 h, 48 h, or 28 days of reperfusion. Aged male and female mice (18-24 months) were subjected to permanent ischemic stroke and sacrificed 48 h later. Infarct volumes were calculated using TTC staining (24-48 h) or Cresyl violet staining (28d). Sensorimotor tests (weight grip, vertical grid, and open field) were performed at indicated timepoints. Blood-brain barrier (BBB) damage, tight junction proteins, matrix metalloproteinase-9 (MMP-9), and neuroinflammatory markers were assessed via immunoblotting, ELISA, immunohistochemistry, and RT-qPCR. Differential gene expression profiles were generated through bulk RNA sequencing and nanoString®. RESULTS: Global genetic deletion of Ripk2 resulted in decreased infarct sizes and reduced neuroinflammatory markers 24 h after stroke compared to wild-type controls. Ripk2 global deletion also improved both acute and long-term behavioral outcomes with powerful effects on reducing infarct volume and mortality at 28d post-stroke. Conditional deletion of microglial Ripk2 (mKO) partially recapitulated our results in global Ripk2 deficient mice, showing reductive effects on infarct volume and improved behavioral outcomes within 48 h of injury. Finally, bulk transcriptomic profiling and nanoString data demonstrated that Ripk2 deficiency in microglia decreases genes associated with MAPK and NF-κB signaling, dampening the neuroinflammatory response after stroke injury by reducing immune cell activation and peripheral immune cell invasion. CONCLUSIONS: These results reveal a hitherto unknown role for RIPK2 in the pathogenesis of ischemic stroke injury, with microglia playing a distinct role. This study identifies RIPK2 as a potent propagator of neuroinflammatory signaling, highlighting its potential as a therapeutic target for post-stroke intervention.

Design, synthesis and biological evaluation of 4-aminoquinoline derivatives as receptor-interacting protein kinase 2 (RIPK2) inhibitors.

Receptor-interacting protein kinase 2 (RIPK2) is an essential protein kinase mediating signal transduction by NOD1 and NOD2, which play an important role in regulating immune signalling. In this study, we designed and synthesised a novel series of 4-aminoquinoline-based derivatives as RIPK2 inhibitors. In vitro, compound 14 exhibited high affinity (IC50 = 5.1 ± 1.6 nM) and excellent selectivity to RIPK2 showing in a dendrogram view of the human kinome phylogenetic tree. Bearing favourable lipophilicity and eligible lipophilic ligand efficiency (LipE), compound 14 was selected to investigate cellular anti-inflammatory effect and was identified as a potent inhibitor to reduce the secretion of MDP-induced TNF-α with a dose-dependent manner. Moreover, compound 14 showed moderate stability in human liver microsome. Given these promising results, compound 14 could serve as a favourable inhibitor of RIPK2 for further physiological and biochemical research so as to be used in therapeutic treatment.

Osthole, an ingredient from Cnidium monnieri, reduces the pyroptosis and apoptosis in bronchial epithelial cells.

Osthole is the prominent active ingredient isolated from Cnidium. The role of osthole in chronic obstructive pulmonary disease (COPD) was investigated herein. Bronchial epithelial 16HBE cells were exposed to cigarette smoke extract (CSE) to generate injury models. The concentration of CSE had an inverse correlation with cell viability. Osthole suppressed inflammation, oxidative stress, apoptosis, and pyroptosis in 16HBE cells, along with a decrease in RIPK2 level. RIPK2 overexpression reversed the effects of osthole on the abovementioned aspects. This study found that the osthole could reduce RIPK2 level, inhibit pyroptosis, and alleviate the damage in 16HBE cells under CSE stimulation.

Identification and expression profiling of receptor-interacting serine/threonine-protein kinase 2 in Siberian sturgeon (Acipenser baerii).

Receptor-interacting serine/threonine-protein kinase 2 (RIPK2) is an adaptor protein of the pattern recognition receptors NOD1 and NOD2 involved in regulating inflammatory response and resisting pathogenic microbial infection. In this study, Acipenser baerii RIPK2 (AbRIPK2) was identified. The open reading frame of AbRIPK2 was 1815 bp encoding 604 amino acids. AbRIPK2 possessed the typical N-terminal kinase domain (KD) and C-terminal caspase recruitment domain (CARD). The phylogenetic tree analysis revealed that AbRIPK2 shared a relatively high identity with bony fish. Real-time fluorescence quantitative PCR (qRT-PCR) results indicated that AbRIPK2 was highly expressed in the gill, followed by muscle, liver and heart. AbRIPK2 was significantly induced in the spleen and valvular intestine after Streptococcus iniae and Aeromonas hydrophila infection. AbRIPK2 was significantly upregulated after peptidoglycan (PGN) treatment in the splenic leukocytes. This study indicated that AbRIPK2 played a potential role in resisting the pathogenic infection of Siberian sturgeon by responding to bacteria.

Novel Biomarkers for Inflammatory Bowel Disease and Colorectal Cancer: An Interplay between Metabolic Dysregulation and Excessive Inflammation.

Persistent inflammation can trigger altered epigenetic, inflammatory, and bioenergetic states. Inflammatory bowel disease (IBD) is an idiopathic disease characterized by chronic inflammation of the gastrointestinal tract, with evidence of subsequent metabolic syndrome disorder. Studies have demonstrated that as many as 42% of patients with ulcerative colitis (UC) who are found to have high-grade dysplasia, either already had colorectal cancer (CRC) or develop it within a short time. The presence of low-grade dysplasia is also predictive of CRC. Many signaling pathways are shared among IBD and CRC, including cell survival, cell proliferation, angiogenesis, and inflammatory signaling pathways. Current IBD therapeutics target a small subset of molecular drivers of IBD, with many focused on the inflammatory aspect of the pathways. Thus, there is a great need to identify biomarkers of both IBD and CRC, that can be predictive of therapeutic efficacy, disease severity, and predisposition to CRC. In this study, we explored the changes in biomarkers specific for inflammatory, metabolic, and proliferative pathways, to help determine the relevance to both IBD and CRC. Our analysis demonstrated, for the first time in IBD, the loss of the tumor suppressor protein Ras associated family protein 1A (RASSF1A), via epigenetic changes, the hyperactivation of the obligate kinase of the NOD2 pathogen recognition receptor (receptor interacting protein kinase 2 [RIPK2]), the loss of activation of the metabolic kinase, AMP activated protein kinase (AMPKα1), and, lastly, the activation of the transcription factor and kinase Yes associated protein (YAP) kinase, that is involved in proliferation of cells. The expression and activation status of these four elements are mirrored in IBD, CRC, and IBD-CRC patients and, importantly, in matched blood and biopsy samples. The latter would suggest that biomarker analysis can be performed non-invasively, to understand IBD and CRC, without the need for invasive and costly endoscopic analysis. This study, for the first time, illustrates the need to understand IBD or CRC beyond an inflammatory perspective and the value of therapeutics directed to reset altered proliferative and metabolic states within the colon. The use of such therapeutics may truly drive patients into remission.

Integrated analysis of mRNA and microRNA expression pattern reveals differential transcriptome signatures in RIPK2 over-expressing chicken macrophages infected with avian pathogenic E. coli.

1. As RIPK2 (receptor interacting serine/threonine kinase 2) has been shown to to alleviate excessive inflammatory responses, the following study conducted a systematic and in-depth analysis of the mRNA-seq and miRNA-seq data from chicken macrophages with/without over-expression of RIPK2 (oeRIPK2) combined with/without avian pathogenic E. coli (APEC) infection to identify the miRNA-mRNA interaction network and potential signalling pathways involved.2. A total of 9,201 differentially expressed (DE) mRNAs and 300 DE miRNA were identified in both oeRIPK2+APEC vs. APEC and oeRIPK2 vs. the wild-type (WT). Moreover, 4,269 instances of co-expression between miRNAs and mRNAs were seen involving 1,652 DE mRNAs and 164 DE miRNAs.3. Functional analysis of the DE mRNAs in the miRNA-mRNA interaction network showed that 223 biological processes and five KEGG pathways were significantly enriched in the two comparisons. In total, 128 pairs of miRNA-mRNA interactions were involved in the identified MAPK signalling pathway and focal adhesion immune related pathways.4. Significantly, these screened miRNAs (gga-miR-222b-5p and gga-miR-214) and their target genes were highly correlated with APEC infection and RIPK2. These recognised key genes, miRNA and the overall miRNA-mRNA regulatory network, enables better understanding of the molecular mechanism of host response to APEC infection, especially related to RIPK2.

Small molecule inhibitors reveal an indispensable scaffolding role of RIPK2 in NOD2 signaling.

RIPK2 mediates inflammatory signaling by the bacteria-sensing receptors NOD1 and NOD2. Kinase inhibitors targeting RIPK2 are a proposed strategy to ameliorate NOD-mediated pathologies. Here, we reveal that RIPK2 kinase activity is dispensable for NOD2 inflammatory signaling and show that RIPK2 inhibitors function instead by antagonizing XIAP-binding and XIAP-mediated ubiquitination of RIPK2. We map the XIAP binding site on RIPK2 to the loop between ß2 and ß3 of the N-lobe of the kinase, which is in close proximity to the ATP-binding pocket. Through characterization of a new series of ATP pocket-binding RIPK2 inhibitors, we identify the molecular features that determine their inhibition of both the RIPK2-XIAP interaction, and of cellular and in vivoNOD2 signaling. Our study exemplifies how targeting of the ATP-binding pocket in RIPK2 can be exploited to interfere with the RIPK2-XIAP interaction for modulation of NOD signaling.

Pan-cancer analysis reveals RIPK2 predicts prognosis and promotes immune therapy resistance via triggering cytotoxic T lymphocytes dysfunction.

BACKGROUND: Receptor-interacting protein kinase 2 (RIPK2, also known as RIP2) was reported to be associated with bacterial infections as well as inflammatory responses. However, the role of RIPK2 in prognosis and immunotherapy response is yet to be elucidated in human pan-cancer. METHODS: In this study, we investigated the expression, gene alteration landscape and prognostic value of RIPK2 in 33 cancers through various databases including Ualcan, cBioportal and Gene Expression Profiling Interactive Analysis 2 (GEPIA2). Then, the correlation between RIPK2 and immune infiltration, immune score, stromal score, and ESTIMATE score was investigated in the Cancer Genome Atlas (TCGA) and tumor immune estimation resource (TIMER) databases. Independent cohorts were utilized to explore the role of RIPK2 in tumor immunotherapy response. Furthermore, Gene set enrichment analysis (GSEA) was conducted to explore the mechanisms by which RIPK2 regulates immune therapy resistance. Single-cell RNA-seq datasets were used to analyze the expression level of RIPK2 on different immune cells. Moreover, CellMiner database was used to explore the relationship between RIPK2 expression with drug response. RESULT: Compared with normal tissue, tumor tissue had a higher expression level of RIPK2 in various cancers. Survival analysis showed that high expression of RIPK2 associated with poor prognosis in numerous cancers. RIPK2 was found to promote a series of immune cell infiltration and B cells, macrophages, and neutrophils were significantly positively correlated with the expression of RIPK2. Moreover, RIPK2 affected immune score, stromal score and ESTIMATE score for a wide range of cancers. In the vast majority of 33 cancers, gene co-expression analysis showed that RIPK2 was positively correlated with the expression of immune checkpoint markers, such as PDCD1 (PD-1), CD274 (PD-L1), CTLA4 and TIGIT. RIPK2 aggravated cytotoxic T lymphocyte (CTL) dysfunction and related to the poor efficacy of immune checkpoint blockade in skin cutaneous melanoma (SKCM) and kidney renal clear cell carcinoma (KIRC). High expression of RIPK2 promoted innate immunotherapy resistance and adaptive immunotherapy resistance through IL-6/JAK/STAT3 signaling, interferon-gamma response, and interferon-alpha response pathway. CONCLUSIONS: These results confirmed that RIPK2 could serve as a prognostic biomarker and promoted immune therapy resistance via triggering cytotoxic T lymphocytes dysfunction.

A regulatory region on RIPK2 is required for XIAP binding and NOD signaling activity.

Signaling via the intracellular pathogen receptors nucleotide-binding oligomerization domain-containing proteins NOD1 and NOD2 requires receptor interacting kinase 2 (RIPK2), an adaptor kinase that can be targeted for the treatment of various inflammatory diseases. However, the molecular mechanisms of how RIPK2 contributes to NOD signaling are not completely understood. We generated FLAG-tagged RIPK2 knock-in mice using CRISPR/Cas9 technology to study NOD signaling mechanisms at the endogenous level. Using cells from these mice, we were able to generate a detailed map of post-translational modifications on RIPK2. Similar to other reports, we did not detect ubiquitination of RIPK2 lysine 209 during NOD2 signaling. However, using site-directed mutagenesis we identified a new regulatory region on RIPK2, which dictates the crucial interaction with the E3 ligase XIAP and downstream signaling outcomes.

Design, synthesis, and structure-activity relationship of novel RIPK2 inhibitors.

The NOD1/2 (nucleotide-binding oligomerization domain-containing protein 1/2) signaling pathways are involved in innate immune control and host defense. NOD dysfunction can result in a variety of autoimmune disorders. NOD-induced generation of inflammatory cytokines is mediated by receptor-interacting protein kinase 2 (RIPK2), which has been considered as a promising therapeutic target. Herein, we disclose the design, synthesis, and SAR study of a series of RIPK2 inhibitors. The lead compound 17 displayed a high affinity for RIPK2 (Kd = 5.9 nM) and was capable of inhibiting RIPK2 kinase function in an ADP-Glo assay. In vitro DMPK studies showed that compound 17 had good metabolic stability and no CYP inhibition. Compound 17 effectively suppressed inflammatory cytokine production in both cells and animal model.

The protective effects of natural product tunicatachalcone against neuroinflammation via targeting RIPK2 in microglia BV-2 cells stimulated by LPS.

Microglia-induced neuroinflammation plays a critical role in neurological diseases. At present, RIPK2 is considered to participate in inflammatory and autoimmune cellular pathways and diseases. RIPK2 is found to be a pivotal therapeutic target in neurologic disorders related to inflammation. In our research, we discovered the protective function of tunicatachalcone (TC) against neuroinflammation. TC is a natural chalcone compound derived from Pongamia pinnata, a medicinal plant. The results revealed that TC (5-20 µM) ameliorated the activation of BV-2 microglia induced by lipopolysaccharide (LPS) in a dose-dependent way, which was proved by the reduced production of inflammation-related mediators. By using SPR-LC-MS/MS analysis, we revealed the potent inhibitory function of TC against neuroinflammation mediated by microglia via targeting RIPK2. A strong binding between TC and RIPK2 was further demonstrated based on the results of SPR, MST and molecular modeling. Through applying mRNA transcriptomics and bioinformatics analysis, it was demonstrated that TC could mediate RIPK2-dependent gene transcription to exert the neuroprotective effect. In summary, our research presented that RIPK2 was a possible therapeutic target of TC.